title: Two-photon fluorescence imaging of intracellular hydrogen peroxide with chemoselective fluorescent probes.
abstract: We present the application of two-photon fluorescence (TPF) imaging to monitor intracellular hydrogen peroxide (H₂O₂) production in brain cells. For selective imaging of H₂O₂ over other reactive oxygen species, we employed small-molecule fluorescent probes that utilize a chemoselective boronate deprotection mechanism. Peroxyfluor-6 acetoxymethyl ester detects global cellular H₂O₂ and mitochondria peroxy yellow 1 detects mitochondrial H₂O₂. Two-photon absorption cross sections for these H₂O₂ probes are measured with a mode-locked Ti:sapphire laser in the wavelength range of 720 to 1040 nm. TPF imaging is demonstrated in the HT22 cell line to monitor both cytoplasmic H₂O₂ and localized H₂O₂ production in mitochondria. Endogenous cytoplasmic H₂O₂ production is detected with TPF imaging in rat astrocytes modified with d-amino acid oxidase. The TPF H₂O₂ imaging demonstrated that these chemoselective probes are powerful tools for the detection of intracellular H₂O₂.
authors.affiliation: University of Maryland, Fischell Department of Bioengineering, College Park, Maryland 20742bWeill Medical College of Cornell University, Burke Medical Research Institute, White Plains, New York 10605.
authors.forename: Hengchang
authors.initials: H
authors.lastname: Guo
authors.forename: Hossein
authors.initials: H
authors.lastname: Aleyasin
authors.forename: Scott S
authors.initials: SS
authors.lastname: Howard
authors.forename: Bryan C
authors.initials: BC
authors.lastname: Dickinson
authors.forename: Vivian S
authors.initials: VS
authors.lastname: Lin
authors.forename: Renee E
authors.initials: RE
authors.lastname: Haskew-Layton
authors.forename: Chris
authors.initials: C
authors.lastname: Xu
authors.forename: Yu
authors.initials: Y
authors.lastname: Chen
authors.forename: Rajiv R
authors.initials: RR
authors.lastname: Ratan
mesh_terms: D000818:Animals; D005456:Fluorescent Dyes / Q000032:analysis* / Q000737:chemistry; D006861:Hydrogen Peroxide / Q000032:analysis*; D042541:Intracellular Space / Q000737:chemistry; D008856:Microscopy, Fluorescence / Q000379:methods*; D057054:Molecular Imaging / Q000379:methods*; D061848:Optical Imaging; D018384:Oxidative Stress; D051381:Rats; D017207:Rats, Sprague-Dawley
chemical_list: D005456:Fluorescent Dyes; D006861:Hydrogen Peroxide